Cordyceps militaris extract stimulates Cl(-) secretion across human bronchial epithelia by both Ca(2+)(-) and cAMP-dependent pathways

Ethnopharmacological relevance

The caterpillar fungus Cordyceps militaris (CM; Clavicipitaceae) is a well-known traditional Chinese medicine that can be artificially cultivated on a large scale. We have previously demonstrated that its stimulatory action on ion transport in human airway epithelia is similar to Cordyceps sinensis (Clavicipitaceae), which has been traditionally used to treat respiratory diseases.

Aim of the study

To investigate the signal transduction mechanism(s) underlying CM-induced ion transport activity in cultured human bronchial epithelia.

Materials and methods

16HBE14o-, a human bronchial epithelial cell line, was used to study the regulation of ion transport by the water extract of CM. CM extract was added to the apical or basolateral aspect of the epithelia. In subsequent experiments, different Cl⁻ channel and K⁺ channel blockers, adenylate cyclase and protein kinase A (PKA) inhibitors, and an intracellular Ca²⁺ chelator were used to examine the involvement of apical Cl⁻ and basolateral K⁺ channels in mediating CM-induced Cl⁻ secretion and the underlying signal transduction mechanism(s). PKA activity was also measured in 16HBE14o- cells.

Results

CM stimulated Cl⁻ secretion across 16HBE14o- monolayers in a dose-dependent manner. Cl⁻ secretion could be inhibited by apical application of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻channel blocker and the calcium-activated Cl⁻ channel (CaCC) blocker. Cl⁻ secretion was sensitive to basolateral application of different K⁺ channel blockers. Similar inhibitory patterns were obtained in nystatin-permeabilized epithelia. The CM-induced Cl⁻ secretion could be inhibited by adenylate cyclase and PKA inhibitors as well as an intracellular Ca²⁺ chelator. Data from the PKA assay suggested that CM extract caused a significant increase in PKA activity compared with untreated control epithelia.

Conclusions

These results suggest that CM extract stimulated Cl⁻ secretion across human bronchial epithelia, possibly via apical CFTR and CaCC, and the basolateral K⁺ channels are involved in driving apical Cl⁻ exit. The underlying signal transduction mechanisms involve both cAMP- and Ca²⁺-dependent pathways.     

左归丸对去势大鼠钙转运通路相关蛋白的影响

目的:探讨左归丸对去势大鼠骨密度和骨组织中钙转运通路相关蛋白的影响。方法:选取48只雌性SD大鼠,随机分为正常组,假手术组,模型组,尼尔雌醇组(0.167 mg·kg^-1),左归丸低、高剂量组(9.6,38.4 g·kg^-1)。除假手术组和正常组,其余组切除大鼠卵巢,造模成功后3个月进行各组干预。3个月后处死大鼠,检测大鼠骨密度(BMD)和骨代谢标志物;蛋白免疫印迹法(Western blot)检测钙转运通路(骨组织)瞬时受体电位通道V5(TRPV5),钙结合蛋白-D28K(CaBP-D28K),钠钙交换器1(NCX1)和细胞膜钙汞(PMCA1b)蛋白表达。结果:与正常组比较,模型组血钙降低(P<0.01),与模型组比较,左归丸高剂量组和尼尔雌醇组大鼠血钙显著升高(P<0.01);与正常组比较,模型组BMD显著降低(P<0.01),与模型组比较,左归丸高剂量组与尼尔雌醇组大鼠BMD均有改善(P<0.05);与假手术组和正常组比较,模型组各蛋白表达均有不同程度上调(P<0.05,P<0.01),与模型组比较,左归丸高剂量组中TRPV5蛋白表达有显著下调(P<0.01),各干预组中的CaBP-D28K与NCX1蛋白表达均有不同程度的下调(P<0.05,P<0.01),PMCA1b蛋白表达在左归丸低剂量组和尼尔雌醇组内表达均有不同程度下调(P<0.05,P<0.01)。结论:左归丸可不同程度的降低大鼠破骨细胞中TRPV5,CAPB-D28K,PMCA1b,NCX1等介导破骨细胞吸收钙离子的重要通道蛋白的表达,左归丸的抗骨质疏松机理有可能是通过抑制TRPV5介导的破骨细胞的骨吸收而起作用。     

Vasodilatory effects of ethanol extract of Radix Paeoniae Rubra and its mechanism of action in the rat aorta

Ethnopharmacological relevance

Radix Paeoniae Rubra (RPR) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China. RPR is the radix of either Paeonia lactiflora Pall. or Paeonia veitchii Lynch. RPR has a wide variety of pharmacological actions such as anti-thrombus, anti-coagulation, and anti-atherosclerotic properties, protecting heart and liver. However, the mechanisms involved are to be defined.

Aim of the study

The aim of the present study was to define the effect of Paeonia lactiflora Pall. extracts on vascular tension and responsible mechanisms in rat thoracic aortic rings.

Materials and methods

Ethanol extract of Paeonia lactiflora Pall. (EPL) was examined for their vascular relaxant effects in isolated phenylephrine-precontracted rat thoracic aorta.

Results

EPL induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Vascular relaxation induced by EPL was significantly inhibited by removal of the endothelium or pretreatment of the rings with N^G-nitro-l-arginine methylester (l-NAME) or 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ). Extracellular Ca²⁺ depletion or diltiazem significantly attenuated EPL-induced vasorelaxation. Modulators of the store-operated Ca²⁺ entry (SOCE), thapsigargin, 2-aminoethyl diphenylborinate and Gd³⁺, and an inhibitor of Akt, wortmannin, markedly attenuated the EPL-induced vasorelaxation. Further, the EPL-induced vasorelaxation was significantly attenuated by pretreatment with tetraethylammonium, a non-selective K_Ca channels blocker, or glibenclamide, an ATP-sensitive K⁺ channels inhibitor, respectively. Inhibition of cyclooxygenases with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on the EPL-induced vasorelaxation.

Conclusions

The present study suggests that EPL relaxes vascular smooth muscle via endothelium-dependent and Akt- and SOCE-eNOS-cGMP-mediated pathways through activation of both K_Ca and K_ATP channels and inhibition of L-type Ca²⁺ channels.     

A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells

Ethnopharmacological relevance

Entada africana is a plant used in African traditional medicine for the treatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery.

Aims of the study

This study aimed at evaluating the anti-inflammatory activity of fractions of the stem bark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages model.

Materials and methods

The crude extract was prepared using the mixture CH₂Cl₂/MeOH (1:1, v/v) and fractionated by flash chromatography using solvents of increasing polarity to obtain five different fractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitric oxide (NO) production screened by Griess test. The most active fraction was further investigated for its effects on reactive oxygen species (ROS) production using flux cytometry, the expression of inducible nitric oxide synthase (iNOS), pro-and anti-inflammatory cytokines (IL1β, TNFα, IL6, IL10 and IL13) by RT-PCR, and the activity of the enzyme p38 MAPK kinase by enzyme-linked immunosorbent assay (ELISA).

Results

The fractions presented no significant effect on the viability of macrophages at 100 μg/ml after 24 h incubation. The CH₂Cl₂/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NO production with a half inhibition concentration (IC₅₀)=18.36 μg/ml, and showed the highest inhibition percentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 μg/ml. Ea5, as well as Baicalin significantly (P<0.05) inhibited the expression of TNFα, IL6 and IL1β mRNA, attenuated mRNA expression of inducible NO synthase in a concentration-dependent manner, stimulated the expression of anti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPK kinase.

Conclusion

The results of the present study indicate that the fraction Ea5 of Entada africana possesses most potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeutic agent in the treatment of inflammatory related diseases cause by over-activation of macrophages.     

Chemopreventive action of Lygodium flexuosum extract in human hepatoma PLC/PRF/5 and Hep 3B cells

Ethnopharmacological relevance

Lygodium flexuosum (Lygodiaceae), a medicinal fern used in Indian traditional medicine against liver disorders.

Aim of the study

The rationale of the study was to examine whether the n-hexane extract from plant Lygodium flexuosum affects apoptosis on human hepatoma PLC/PRF/5 and Hep 3B cells.

Materials and methods

Chemopreventive activity of the Lygodium flexuosum extract was determined by MTT assay, annexin-V FITC binding to phosphatidyl serine and cleavage of PARP. Subdiploid condition of cells treated with Lygodium flexuosum was analyzed by flow cytometry. Further, used transiently transfected NF-κB reporter in PLC/PRF/5 cells to evaluate the inhibitive effect of Lygodium flexuosum extract.

Results

Lygodium flexuosum extract inhibited the cell viability and induced apoptosis in hepatoma cells in a concentration dependent manner as evidenced by apoptotic changes such as flipping of phosphatidyl serine, cleavage of PARP. Cell cycle analysis showed the subG1 apoptotic population in cells treated with higher concentrations of the extract. When activated with exogenous TNF-α in transfected hepatoma cells it was observed that NF-κB dependent gene expression was inhibited by treatment with Lygodium flexuosum extract in PLC/PRF/5 cells dose-dependently.

Conclusions

This investigation suggests that the Lygodium flexuosum extract has antiproliferative and apoptotic activity in both cancer cells and has inhibitive role in TNF-α induced NF-κB activation in PLC/PRF/5 cells confirms the potential of the extract as a chemopreventive agent.     

The mechanism of vasorelaxation induced by Schisandra chinensis extract in rat thoracic aorta

Aim of the study

Schisandra chinensis (SC) is a known medical herb for the treatment of cardiovascular symptoms associated with menopausal symptoms in Korea. However, the pharmacological action mechanisms involved have not been well studied. This study was aimed to investigate the vascular effects of SC in rat thoracic aorta.

Materials and methods

We isolated the hexane, chloroform, and methanol extracts from SC and evaluated their vasodilatory effects in the rat thoracic aorta.

Results

Hexane extracts of SC (SCHE, 5 × 10⁻⁵ to 10⁻³ g/L) caused a concentration-dependent relaxation in both endothelium-intact and -denuded aortas. The relaxant effect of SCHE on the endothelium-intact aorta was more prominent than on the endothelium-denuded aorta. The former was significantly attenuated by L-NAME, a nitric oxide synthase inhibitor, and ODQ, a soluble guanyl cyclase inhibitor, but not by tetraethylammonium, a nonselective blocker of K⁺ channels, and indomethacin, a cyclooxygenase inhibitor. Furthermore, SCHE caused nitrite production as well as eNOS activation in aortic segments, suggesting implication of NO signal pathway in SCHE-induced relaxation. In endothelium-denuded aorta, SCHE-induced vasorelaxation was also attenuated by calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, but not by ML-9, a MLC kinase inhibitor, suggestive of implication of MLC phosphatase activation. Phenylephrine-enhanced MLC phosphorylation ratio was significantly attenuated by SCHE, which was recovered to the control level by pretreatment with calyculin A.

Conclusions

Taken collectively, these findings suggest that the vascular relaxation evoked by SCHE was mediated by not only endothelium dependent NO pathway but also direct effect on vascular smooth muscle cell via dephosphorylation of MLC.     

Yokukansan, a kampo medicine, protects against glutamate cytotoxicity due to oxidative stress in PC12 cells

Aim of the study

Yokukansan is a traditional Japanese medicine consisted of seven medicinal herbs and has been used for treatment of neurosis, insomnia, and behavioral and psychological symptoms of dementia in Japan. The aim of the present study is to clarify the active compounds responsible for the protective effect of yokukansan against glutamate-induced cytotoxicity in PC12 cells.

Materials and methods

PC12 cells which is a tool for selective evaluation of test substances against oxidative stress was used in the present study. The cell survival rates or glutathione (GSH) levels were evaluated by a MTT reduction assay or GSH assay based on the GSH reductase enzymatic recycling method, respectively.

Results

Glutamate (1-17.5 mM) induced cell death of PC12 cells in a concentration- dependent manner. Yokukansan (125-500 μg/ml) inhibited the glutamate-induced PC12 cell death. When the effects of extracts of the seven constituent herbs in yokukansan on the cell death were examined, Uncaria thorn was found to have the highest potency in the protection. To clarify the active compounds in Uncaria thorn, the effects of seven alkaloids (rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether) on the cell death were further examined. The protective effects were found in hirsutine, hirsuteine, and geissoschizine methyl ether, which also ameliorated the glutamate-induced decrease in GSH levels.

Conclusion

These results suggest that yokukansan protects against PC12 cell death induced by glutamate-mediated oxidative stress, i.e., reduction of intracellular GSH level, and the effect may be mainly attributed to a synergistic effect of the hirsutine, hirsuteine, and geissoschizine methyl ether in Uncaria thorn.     

阿魏酸对人类乳腺癌细胞增殖作用机制的实验研究

目的:考察阿魏酸对人类乳腺癌细胞的增殖作用及可能的作用机制。方法:利用体外培养的ER阳性人乳腺癌细胞系T47D细胞和ER阴性人乳腺癌细胞系MDA-MB-231细胞,通过MTT增殖实验、流式细胞技术检测细胞周期分布、测定ER亚型蛋白表达和用实时荧光定量RT-PCR法检测阿魏酸对雌激素效应基因pS2的调控作用。结果:1×10-5~1×10-7mol.L-1的阿魏酸可促进T47D细胞增殖(P0.05),1×10-8mol.L-1雌激素受体拮抗剂FaslodexTM能抑制这种增殖效应。实验浓度阿魏酸以及它们与1×10-8mol.L-1雌激素受体拮抗剂FaslodexTM共孵育对MDA-MB-231细胞的增殖作用无论在时效或者量效方面均无显著的统计学意义。阿魏酸能使T47D细胞S期细胞数增加,细胞分裂增殖指数升高(P0.05);随着细胞的孵育时间延长至48 h,细胞分裂增殖指数与孵育24 h相比显著降低(P0.01),实验浓度阿魏酸以及它们与1×10-8mol.L-1雌激素受体拮抗剂FaslodexTM共孵育使T47D细胞S期和G2M期细胞比例下降,G0-G1期比例升高,细胞周期停滞于G0-G1期。1×10-7,1×10-6mol.L-1阿魏酸可诱导T47D细胞ERα蛋白表达水平显著升高(P0.01),而对ERβ蛋白表达水平无显著影响。1×10-7,1×10-6mol.L-1阿魏酸均上调T47D细胞雌激素依赖性基因pS2mRNA表达。结论:阿魏酸通过促进ER阳性乳腺癌细胞增殖、上调ERα蛋白和雌激素依赖性基因pS2mRNA表达水平,有植物雌激素样作用。     

Neuroprotective effect of modified Wu-Zi-Yan-Zong granule,a traditional Chinese herbal medicine,on CoCl2-induced PC12 cells

Aim of the study

To investigate the neuroprotective effect of aqueous extract of modified Wu-Zi-Yan-Zong granule (MWG), a traditional Chinese herbal medicine, against CoCl₂-induced neurotoxicity in PC12 cells.

Materials and methods

Cell viability assay, apoptosis rate assay, ROS detection and mitochondrial membrane potential (MMP) assay were performed. In addition, cytochrome c, caspase-3, PARP and MAPKs were also detected by Western blotting.

Results

MWG extract increased viability and suppresses early and middle/late stage apoptosis in a dose-dependent manner in CoCl₂-induced PC12 cells. Moreover, MWG extract decreased the level of intracellular reactive oxygen species (ROS), increased MMP, regulated Bcl-2 family protein expression (Bcl-2 and Bcl-XL) and inhibited the release of cytochrome c from the mitochondria. In addition, MWG extract attenuated activation of caspase-3 and poly ADP-ribose polymerase (PARP) and inhibited the phosphorylation of ERK, c-Jun NH₂-terminal kinase (JNK) and p38 MAPKs.

Conclusions

MWG extract exhibited significant neuroprotective effect on PC12 cells, and this effect may be associated with the suppression of ROS generation and inhibition of mitochondria-mediated caspase and MAPK signaling pathways.     

Phenological and geographical influence in the concentration of selected bioactive 4-phenylcoumarins and chlorogenic acid in Hintonia latiflora leaves

Ethnopharmacological relevance

Hintonia latiflora is a Mexican medicinal plant with well-documented ethnomedical record comprising more than 400 years; in modern Mexico is used for treating several maladies such as diabetes and gastric ulcers. Although the pharmacological actions of the stem-bark and leaves have been demonstrated, the phenological and geographical effect on the concentration of active principles remains unexplored.

Aim of the study

The main goals of this study were to analyze the amount of selected 4-phenylcoumarins and chlorogenic acid in the leaves in order to assess the best harvesting period, and consequently their pharmacological efficacy. In addition, the preclinical antidiabetic efficacy of the infusion of the leaves was corroborated using standard pharmacological tests.

Materials and methods

The aqueous extracts from the leaves of Hintonia latiflora were prepared by infusion. For phenological and geographical comparison, leaves of Hintonia latiflora were collected in two different regions in Chihuahua and Michoacán. The material was analyzed by UPLC applying an analytical method that developed and validated for this purpose following the ICH guidelines. Investigation of the antidiabetic action was accomplished using an acute hypoglycemic test and oral glucose and sucrose tolerance tests.

Results

The validated analytical method was successfully applied for quantifying chlorogenic acid (1) and 4-phenylcoumarins (2–5) in the leaves of 12 different batches (1–12) during one-year period, and seven different batches for each geographical region; the concentration of the metabolites at the phenological cycle was significantly different, their concentration increased during the pre-senescence phase whereas in the leaf renovation stage the highest concentration of 2–5 was reached. The overall analysis of the active compounds concentration between the two populations investigated seems to be less important than the phenological variations. The aqueous extract of the leaves of Hintonia latiflora exerted its antidiabetic effect by different mechanisms showing comparable effect to the organic extract.

Conclusions

The findings of the present investigation reveal that the best harvest season for the leaves of Hintonia latiflora is between the leaves renovation and senescence stages avoiding the flowering period. In addition, no significant differences were found among the two different geographical populations analyzed. The infusions of the leaves, rich in 4-phenylcoumarins and chlorogenic acid, showed comparable antidiabetic action than the organic extract.     

Vascular relaxation by ethanol extract of Xanthoceras sorbifolia via Akt- and SOCE-eNOS-cGMP pathways

Aim of the study

The aim of the present study was to define the effect of Xanthoceras sorbifolia extracts (XS) on vascular tension and responsible mechanisms in rat thoracic aortic rings.

Materials and methods

Ethanol extract of the leaves of XS (EXS) was examined for their vascular relaxant effects in isolated phenylephrine-precontracted rat thoracic aorta.

Results

EXS (0.1-100 μg/ml) induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Endothelium-denudation abolished EXS-induced vasorelaxation. Pretreatment of the endothelium-intact aortic rings with N^G-nitro-l-arginine methylester (l-NAME) and 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ) inhibited EXS-induced vasorelaxation. Inhibition of Ca²⁺ entry via l-type Ca²⁺ channels failed to block the EXS-induced vasorelaxation. Extracellular Ca²⁺ depletion significantly attenuated EXS-induced vasorelaxation. Modulators of the store-operated Ca²⁺ entry (SOCE), thapsigargin, 2-aminoethyl diphenylborinate (2-APB) and Gd³⁺, and an inhibitor of Akt, wortmannin, markedly attenuated the EXS-induced vasorelaxation. EXS increased cGMP levels of the aortic rings in a concentration-dependent manner and the effect was blocked by l-NAME, ODQ, thapsigargin, Gd³⁺, 2-APB, and wortmannin. Further, EXS-induced vasorelaxation was significantly attenuated by tetraethylammonium, a non-selective K_ca channels blocker, but not by glibenclamide, an ATP-sensitive K⁺ channels inhibitor. Inhibition of cyclooxygenase with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on EXS-induced vasorelaxation.

Conclusions

The present study suggests that EXS relaxes vascular smooth muscle via endothelium-dependent NO-cGMP signaling through activation of the Akt- and SOCE-eNOS-sGC pathways, which may, at least in part, be related to the function of K⁺ channels.     

Effect of an extract of Andrographis paniculata leaves on inflammatory and allergic mediators in vitro

Aim of study

Andrographis paniculata has been known to possess widespread traditional application in the treatment of allergy and inflammatory diseases. In the current study, we sought to examine the effects of an extract of Andrographis paniculata leaves on inhibition of lipopolysaccharide (LPS) induced [nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1beta (IL-1 beta), and interleukin-6 (IL-6)] and calcimycin (A23187) induced [leukotriene B₄ (LTB₄), thromboxane B₂ (TXB₂) and histamine] mediators in diverse cell based models.

Materials and methods

Effect of an extract of Andrographis paniculata leaves (AP) was studied on inhibition of LPS induced NO, PGE₂, IL-1 beta and IL-6 in J774A.1 murine macrophages; A23187 induced LTB₄ and TXB₂ in HL-60 promyelocytic leukemic cells and histamine in RBL-2H3 rat basophilic leukemia cells.

Results and conclusion

AP illustrated significant alleviation of pro-inflammatory, inflammatory, and allergic mediators. However, no inhibition was observed against histamine release. This outcome has been summed up to deduce that AP is fairly potent in attenuating the inflammation by inhibiting pro-inflammatory (NO, IL-1 beta and IL-6), inflammatory (PGE₂ and TXB₂) and allergic (LTB₄) mediators.     

Antinociceptive action of ethanolic extract obtained from roots of Humirianthera ampla Miers

Humirianthera ampla Miers is a member of the Icacinaceae family and presents great amounts of di and triterpenoids. These chemical constituents in roots of Humirianthera ampla sustain not only the ethnopharmacological use against snake venom, but also some anti-inflammatory and analgesic properties of the plant. In this study we investigated the antinociceptive action of the ethanolic extract (EE) from roots of the Humirianthera ampla in chemical and thermal models of pain in mice. The oral treatment with ethanolic extract dose-dependently inhibited glutamate-, capsaicin- and formalin-induced licking. However, it did not prevent the nociception caused by radiant heat on the tail-flick test. The ethanolic extract (30 mg/kg) caused marked inhibition of the nociceptive biting response induced by glutamate, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), N-methyl-d-aspartate (NMDA) and substance P. The antinociception caused by ethanolic extract was significantly attenuated by naloxone, l-arginine, WAY100635, ondansetron or ketanserin, but not by caffeine or naloxone methiodide. In conclusion, the ethanolic extract from roots of Humirianthera ampla produces antinociception against neurogenic and inflammatory models of nociception. The mechanisms of antinociception involve nitric oxide, opioid, serotonin and glutamate pathways. Therefore, our results support the ethnopharmacological use of the Humirianthera ampla against inflammatory and painful process caused by snake venom.     

Effect of Rhizoma Polygonati on 12-O-tetradecanoylphorbol-acetate-induced ear edema in mice

Ethnopharmacological relevance

Rhizoma Polygonati is originated from the dried rhizomes of Polygonatum sibircum Red. It has long been used in traditional Chinese medicine for the treatment of inflammatory disorders.

Aim of the study

The present study aims to investigate the anti-inflammatory effect of aqueous extract of Rhizoma Polygonati (ERP) in a mouse model of inflammation induced by 12-O-tetradecanoylphorbol-acetate (TPA).

Materials and methods

The anti-inflammatory effect was evaluated by measuring the ear thickness and activity of myeloperoxidase (MPO). The anti-inflammatory mechanism was explored by determining the protein and mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6.

Results

The results showed that ERP significantly decreased the ear thickness and MPO activity in mouse model of inflammation induced by TPA. In addition, ERP also remarkably inhibited the protein and mRNA levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6.

Conclusions

These results indicate that ERP has potential anti-inflammatory effect on TPA-induced inflammatory in mice, and the anti-inflammatory effect may be mediated, at least in part, by inhibiting the mRNA expression of a panel of inflammatory mediators including iNOS, COX-2, TNF-α, IL-1β, and IL-6.     

外源Ca~(2+)及NO供体硝普钠(SNP)对盐胁迫下紫苏种子萌发及幼苗抗氧化酶活性的影响

目的:通过对紫苏种子萌发及幼苗生理特性的研究,寻找提高紫苏种子及幼苗在盐胁迫条件下抗性能力的途径。方法:测定在外源Ca2+及NO供体硝普钠(SNP)处理后,NaCl胁迫下紫苏种子的发芽势(Gv)、发芽率(Gr)、发芽指数(Gi)和活力指数(Vi),并对紫苏幼苗总生物量以及叶片的丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性进行测定。结果:100 mmol.L-1的NaCl胁迫下的紫苏种子萌发受到显著抑制,但是经过Ca2+和SNP处理后,萌发指标均有升高。外施10 mmol.L-1的Ca2+或0.1 mmol.L-1的SNP都能够有效的缓解NaCl对紫苏种子及幼苗造成的胁迫伤害,经过SNP,Ca2+的复合处理后,效果最为显著,各项指标均达到最大值,发芽势为65.1%,发芽率为89.3%,发芽指数为13.9,活力指数为为0.110 9。复合处理提高了幼苗的总生物量,降低了叶片的MDA含量,显著的提高了SOD,POD,CAT的活性,其结果分别达到0.84,5.71,4.92 U.mg-1,但是乙二醇双(2-氨基乙醚)四乙酸(EGTA)却显著的抑制了SNP对盐害的缓解作用。结论:10 mmol.L-1的Ca2+和0.1 mmol.L-1的SNP的复合处理能够有效的减缓NaCl胁迫对紫苏种子及幼苗产生的伤害,提高了种子及幼苗的抗盐能力。NO可能通过激活紫苏细胞质膜Ca2+通道促进Ca2+的吸收,改变胞内Ca2+浓度来发挥其对NaCl胁迫伤害的缓解作用。     

Ability of certain plant extracts traditionally used to treat ciguatera fish poisoning to inhibit nitric oxide production in RAW 264.7 macrophages

Aim of the study

Ciguatera fish poisoning (CFP) is an intertropical ichthyosarcotoxism that manifests in complex assortment of symptoms in humans. Ciguatoxins (CTXs), issued from Gambierdicus spp., are causative agents of this intoxication. We have recently demonstrated that a Pacific CTX (P-CTX-1B) strongly modulated iNOS expression, leading to overproduction of nitric oxide (NO) in RAW 264.7 murine macrophage cells. NO produced in large amounts is involved in a wide range of pathophysiological processes. Many traditional remedies are commonly used in the Pacific against CFP. In this context, bioassay-guided screening was carried out to study NO inhibiting capacity of 28 selected plant extracts.

Materials and methods

We prepared aqueous extracts of plants used in New Caledonia in the treatment of CFP and screened their NO inhibitory activity in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages.

Results

Among 28 plants tested, Euphorbia hirta (Euphorbiaceae), Syzygium malaccense (Myrtaceae), Schinus terebenthifolius (Anacardiaceae), Punica granatum (Punicaceae), Cerbera manghas (Apocynaceae), Vitex trifolia (Labiateae) and Ximenia americana (Olacaceae) showed inhibitory activity, validating their use as traditional remedies in CFP, and the potential for use in the treatment of conditions accompanied by NO overproduction.

Conclusion

These plants are promising candidates for further screening of their active compounds through activity-guided fractionation.     

Vasorelaxant action of the total alkaloid fraction obtained from Solanum paludosum Moric. (Solanaceae) involves NO/cGMP/PKG pathway and potassium channels

Ethnopharmacological relevance

Solanum paludosum Moric. (jurubeba-roxa) is commonly used to treat hypertension as a substitute for Solanum paniculatum L. (jurubeba verdadeira). The total ethanolic extract from the root bark of Solanum paludosum have been found to cause hypotension in rats.

Aim of the study

To investigate the mechanism by which the total alkaloid fraction obtained from the root bark of Solanum paludosum (FAT-SP) acts as a vasorelaxant agent on rat thoracic aorta.

Materials and methods

Rings of rat aorta were suspended in organ bath containing Krebs solution at 37 °C, bubbled with carbogen mixture (95% O₂ and 5% CO₂) under a resting tension of 1 g. Isometric contractions were measured using a force transducer coupled to an amplifier and a microcomputer.

Results

FAT-SP has been found cause relaxation of the aortic rings pre-contracted with phenylephrine (Phe) in a concentration-dependent manner, in the presence and absence of endothelium. This effect was more potent on the endothelium-intact aorta. In the presence of endothelium, neither indomethacin (non-selective cyclooxygenase inhibitor) nor atropine (non-selective muscarinic receptor antagonist), produced significant changes on the relaxation response. On the other hand, in the presence of calmidazolium (a calmodulin inhibitor), N-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase inhibitor), hydroxocobalamin (HDX) (scavenger of free-radical nitric oxide), 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, selective blocker of soluble guanylate cyclase), Rp-8-bromo-β-phenyl-1,N²-ethenoguanosine 3′:5′-cyclic monophosphorothioate sodium salt hydrate (Rp-8-Br-PET-cGMPS, competitive inhibitor of cGMP-dependent protein kinase G) or TEA⁺ (tetraethylammonium, nonselective potassium channel blocker), the vasorelaxant effect was significantly reduced, suggesting the involvement of NO/sCG/PKG pathway and potassium channel opening in vasorelaxant action of the FAT-SP.

Conclusion

The mechanism of vasorelaxant activity of the FAT-SP on rat aorta involves both NO/sCG/PKG pathway and potassium channels.     

胞内、外Ca2+在水杨酸诱导丹参幼苗迷迭香酸生物合成过程中的作用

目的: 为探讨胞内、外钙离子是否在水杨酸诱导丹参迷迭香酸生物合成中起作用。 方法: 以生长50 d的丹参幼苗为材料,通过喷施水杨酸诱导迷迭香酸合成量增加,再利用胞外钙通道抑制剂verapamil(Vp)和LaCl₃、胞内钙调素拮抗剂trifluoperazine (TFP)和胞内钙通道上IP₃受体抑制剂LiCl处理,分别考察水杨酸、钙离子通道抑制剂/钙调素拮抗剂处理后迷迭香酸的合成量及其合成相关酶(PAL,TAT)的活性变化。 结果: 2.0 mmol·L^-1的水杨酸处理24 h后可诱导迷迭香酸合成量升高到(40.51±2.16) mg·g^-1,是对照的1.97倍,迷迭香酸合成相关酶PAL活性升高到对照的1.42倍,TAT活性升高到对照的1.29倍;Vp,LaCl₃,TFP,LiCl处理均抑制了水杨酸诱导的PAL,TAT活性的升高,从而导致了迷迭香酸合成量降低。 结论: 水杨酸诱导丹参迷迭香酸生物合成过程中,胞内、外钙离子发挥着重要的信号转导作用。     

Rosmarinus officinalis polyphenols activate cholinergic activities in PC12 cells through phosphorylation of ERK1/2

Aim of the study

This paper aimed to elucidate the traditional use of Rosmarinus officinalis through the investigation of cholinergic activities and neuronal differentiation in rat pheochromocytoma PC12 cells. These effects were examined in relation to the plant's habitat, the extraction procedure, and the major active compounds of R. officinalis.

Materials and methods

Cell viability, cell differentiation, acetylcholinesterase (AChE) activity, total choline, acetylcholine (ACh) and extracellular signal-regulated kinases (ERK1/2) were determined in PC12 cells treated with extracts and HPLC-identified polyphenols of R. officinalis originated from Tunisian semi-arid and subhumid area in comparison with nerve growth factor (NGF).

Results

R. officinalis extracts potentiated cell differentiation and significantly enhanced AChE activity in PC12 cells. The highest AChE activity was induced by semi-arid hydro-ethanolic extract (137% of control). Among HPLC-identified and screened polyphenols, carnosic acid (CA) and rosmarinic acid (RA) significantly induced cell differentiation, increased ACh level, and enhanced AChE activity in PC12 cells. U0126, inhibitor of ERK1/2, significantly reduced CA and RA effects on cell differentiation and AChE activity.

Conclusions

R. officinalis’ CA and RA exhibited neurotrophic effects in PC12 cells through cell differentiation induction and cholinergic activities enhancement. These effects could be regulated by mitogen-activated protein kinase (MAPK), ERK1/2 signaling pathway.